Bisphosphocin gel formulations and uses thereof

ABSTRACT

Gel formulations having antimicrobial activity are disclosed. Methods of using the gel formulation are further disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/518,262 filed on Jun. 12, 2017, which is incorporated byreference herein in its entirety.

FIELD

The present disclosure relates generally to gel formulations ofdeoxyribose-based therapeutics. The formulations of some embodiments ofthe present disclosure are particularly useful for the topical treatmentof microbial infections.

BACKGROUND

Pathogenic microbial infections of the skin and other soft tissues causea wide range of diseases. Epicutaneous bacterial infections are ofparticular concern and are responsible for a large number of seriousdiseases. The increasingly widespread prevalence of antibiotic-resistantbacterial strains, such as methicillin-resistant Staphylococcus aureus(MRSA), poses a growing health threat in the United States and elsewherein the world. Despite significant efforts over the past few decades, thediscovery of new antibiotics has proven to be exceedingly difficult,resulting in a steady decline in viable therapeutic options as bacteriabecome more resistant to existing antibiotics.

The treatment of Gram-negative bacterial infections is particularlychallenging due to the lack of efficacious agents and the increasingincidence of drug resistant strains. Gram-negative bacteria are known tocause serious and sometimes life-threatening skin and soft tissueinfections (SSTIs) including wound or surgical site infections.Gram-negative bacteria are often resistant to most or all of thecurrently available drugs.

Biofilms may also play a role in the most serious SSTIs. Biofilms arecurrently estimated to be responsible for over 65% of nosocomialinfections and 80% of all microbial infections. Treatingbiofilm-mediated infections has proven to be very difficult because thetypical minimum inhibitory concentrations (MICs) and the minimumbactericidal concentrations (MBCs) for biofilm bacterial cells aretypically 10-1000 times higher than planktonic bacterial cells.

A new class of antibiotics called “Bisphosphocins” has been discoveredthat may offer an approach to treating a wide range of clinicallyimportant bacterial infections, including those caused bymultidrug-resistant bacteria. Members of the Bisphosphocin class arecharacterized by a core deoxyribose unit having two protected phosphategroups. These molecules are highly protonated/acidified and exhibitexcellent chemical stability, low pH resistance, and resistance todegradation by nucleases. The class of Bisphosphocins has been describedas having antimicrobial activity. As antibiotics, it is believed thatBisphosphocins act by rapidly disrupting the bacterial cell membraneand/or cell wall in many different bacterial strains, includingGram-positive, Gram-negative, and certain antibiotic resistant strains.The most advanced member of the Bisphosphocin class, Nu-3, is currentlyundergoing clinical trials for treating infected diabetic foot ulcers.

The formulation of Nu-3 currently being used in clinical trials is anaqueous solution. Use of this formulation to treat diabetic foot ulcershas proven to be challenging. A more adherent topical formulation ofNu-3 and other Bisphosphocins would be desirable for several reasons. Amore viscous formulation, like a gel, in particular, would be easier andmore convenient to apply, aesthetically pleasing, and would allow forlonger topical exposure times. Preparing a gel formulation for theBisphosphocin class of compounds has been particularly difficult forseveral reasons. Because of their reduced level of activity whenformulated at higher (more basic) pH levels, Bisphosphocins and theircorresponding salts are typically formulated at highly acidic pH in therange of pH 1 to pH 5. For example, Bisphosphocins such as Nu-3 need tobe formulated at pH 2 or below to retain the most potent antimicrobialactivity.

In addition, topical formulations containing relatively highconcentrations of the active Bisphosphocin compounds are desired buthave also been difficult to make. Additionally, because theBisphosphocins and their salts are strongly ionic, it has been difficultto use ionic polymers or emulsifying agents with these compounds. Thus,the resulting formulations are unstable and display poor cohesiveproperties.

Therefore, there is a need for new antimicrobial Bisphosphocinformulations that overcome the difficulties described above.

SUMMARY

The present disclosure provides gel formulations and uses thereof thataddress the challenges detailed above.

An aspect of the present disclosure provides a gel formulation. In someembodiments, the gel formulation includes a Bisphosphocin selected fromthe group consisting of Nu-2, Nu-3, Nu-4, Nu-5, and Nu-8, or apharmaceutically acceptable salt thereof, or any combination thereof; afatty alcohol thickening agent; and a nonionic polymer emulsifier. Insome embodiments, the Bisphosphocin is Nu-3, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the formulation has a pH of about pH 1 to about pH5. In some embodiments, the formulation has a pH of about pH 1.5 toabout pH 4. In some embodiments, the formulation has a pH of about pH 3to about pH 4.

In some embodiments, the Bisphosphocin is present in the formulation inan amount from about 1% to about 20% (weight/weight). In someembodiments, the Bisphosphocin is present in the formulation in anamount from about 5% to about 15% (weight/weight). In some embodiments,the Bisphosphocin is present in the formulation in an amount from about30% to about 50% (weight/weight).

In some embodiments, the fatty alcohol thickening agent is present inthe formulation in an amount from about 1% to about 50% (weight/weight).In some embodiments, the fatty alcohol thickening agent is present inthe formulation in an amount from about 1% to about 20% (weight/weight).In some embodiments, the fatty alcohol thickening agent is present inthe formulation in an amount from about 1% to about 10% (weight/weight).

In some embodiments, the nonionic polymer emulsifier is present in theformulation in an amount from about 0.25% to about 15% (weight/weight).In some embodiments, the nonionic polymer emulsifier is present in theformulation in an amount from about 0.5% to about 5% (weight/weight).

In some embodiments, the formulation further comprises a diluentselected from the group consisting of water, glycerol, mannitol, saline,and phosphate buffered saline. In some embodiments, the diluent iswater. In some embodiments, the water is present in the formulation inan amount from about 65% to about 97.5% (weight/weight).

In some embodiments, the fatty alcohol thickening agent is selected fromthe group consisting of cetyl alcohol, lauryl alcohol, stearyl alcohol,cetosteryl alcohol, myristyl alcohol, docosanol alcohol, and oleylalcohol. In some embodiments, the fatty alcohol thickening agent iscetosteryl alcohol. In some embodiments, the cetosteryl alcohol ispresent in the formulation in an amount from about 2% to about 10%(w/w).

In some embodiments, the nonionic polymer is selected from the groupconsisting of ceteth-20, steareth-20, and ceteareth-20. In someembodiments, the nonionic polymer emulsifier is ceteareth 20. In someembodiments, the ceteareth-20 is present in the formulation in an amountgreater than about 0.5% (w/w). In some embodiments, the ceteareth-20 ispresent in the formulation in an amount of about 0.5% to about 5% (w/w).

In some embodiments, the formulation is adapted for topicaladministration.

Another aspect of the present disclosure provides a method of treatingan infection in a patient in need thereof, the method comprisingadministering an effective amount of a formulation of the presentdisclosure to the patient. In some embodiments, the infection is aninfection of a diabetic foot ulcer. In some embodiments, the infectionis an infection of a burn wound. In some embodiments, the infection isan infection of a complicated venous leg ulcer. In some embodiments, theinfection is an otitis externa infection.

Another aspect of the present disclosure provides a method of treatingacne vulgaris in a patient in need thereof, the method comprisingadministering an effective amount of a formulation of the presentdisclosure to the patient.

Another aspect of the present disclosure provides a method of treatingotitis externa in a patient in need thereof, the method comprisingadministering an effective amount of a formulation of the presentdisclosure to the patient.

Another aspect of the present disclosure provides a method of treatingonychomycosis in a patient in need thereof, the method comprisingadministering an effective amount of a formulation of the presentdisclosure to the patient.

Another aspect of the present disclosure provides a method of treatingconjunctivitis in a patient in need thereof, the method comprisingadministering an effective amount of a formulation of the presentdisclosure to the patient.

In some embodiments, the patient is administered at least one additionalactive ingredient. In some embodiments, the patient is a human.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in therapy.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of an infection.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of an infection of adiabetic foot ulcer.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of an infection in a burnwound.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of an infection of acomplicated venous leg ulcer.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of acne vulgaris.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of otitis externa.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of onychomycosis.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the treatment of conjunctivitis.

Another aspect of the present disclosure provides a formulation of thepresent disclosure for use in the manufacture of a medicament. In someembodiments, the medicament is for treating an infection in a burnwound. In some embodiments, the medicament is for treating an infectionin a diabetic foot ulcer. In some embodiments, the medicament is fortreating an infection in a complicated venous leg ulcer. In someembodiments, the medicament is for treating acne vulgaris. In someembodiments, the medicament is for treating otitis externa. In someembodiments, the medicament is for treating onychomycosis. In someembodiments, the medicament is for treating conjunctivitis. In someembodiments, the medicament is adapted for topical administration.

One advantage of a formulation according to an embodiment of the presentdisclosure is that the mechanism of action of the activity of theBisphosphocins is effective against many different clinically relevantpathogenic bacteria, including both gram positive and gram negativebacteria. Another advantage of a formulation according to an embodimentof the present disclosure is that such formulation of an embodiment isnon-toxic to a patient treated with an effective amount of theformulation of the present disclosure.

A further advantage of a formulation according to an embodiment of thepresent disclosure is that such formulation is useful for treatinginfections caused by biofilms. Another advantage of a formulationaccording to an embodiment of the present disclosure is that theformulation may be administered in an effective amount to treat apatient suffering from a dermatological disorder, an ophthalmiccondition, or a wound. Another advantage of a formulation according toan embodiment of the present disclosure is that such formulation can beused during or after surgery (for example, in connection with surgicalincisions or implants).

A further advantage of a formulation according to an embodiment of thepresent disclosure is that such formulation may be administered in aneffective amount to treat a patient suffering from a dermatologicaldisorder (such as, for example, complicated skin and skin structureinfections (cSSSI), acne vulgaris, otitis externa, or onychomycosis), anophthalmic condition (such as, for example, conjunctivitis (pink eye),or a wound (for example, burn wounds, complicated diabetic foot ulcers(cDFCU) or complicated venous leg ulcers (cVLU)).

These and other objects, advantages, and features of the presentdisclosure will become apparent to those skilled in the art upon readingthe details of the compounds and formulations according to the presentdisclosure and uses thereof as more fully described below.

DETAILED DESCRIPTION

The embodiments described below are not intended to be exhaustive or tolimit the invention to the precise forms disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may appreciate and understand theprinciples and practices of this disclosure.

The present disclosure provides a gel formulation. In some embodiments,the gel formulation includes a Bisphosphocin selected from the groupconsisting of Nu-2, Nu-3, Nu-4, Nu-5, and Nu-8, or a pharmaceuticallyacceptable salt thereof, or any combination thereof. As used herein, theterm “Bisphosphocin” refers to a class of chemical compounds havingantimicrobial activity, including Nu-2, Nu-3, Nu-4, Nu-5, and Nu-8, or apharmaceutically acceptable salt thereof. U.S. Pat. Nos. 6,627,215,6,211,162, 7,868,162, 7,176,191, 8,435,960, and 6,211,349, all of whichare hereby incorporated by reference in their entireties, discloseBisphosphocins and how to make and use Bisphosphocins.

As used herein, the term “formulation” refers to a pharmaceuticalpreparation that contains a Bisphosphocin, or a pharmaceuticallyacceptable salt thereof, and is suitable for administration to a patientfor therapeutic purposes. As used herein, the term “patient” refers to aliving organism that is treated with a Bisphosphocin, including withoutlimitation any mammal such as, for example, humans, other primates(e.g., monkeys, chimpanzees, etc.), companion animals (e.g., dogs, cats,horses, etc.), farm animals (e.g., goats, sheep, pigs, cattle, etc.),laboratory animals (e.g., mice, rats, etc.), and wild and zoo animals(e.g., wolves, bears, deer, etc.). As used herein, the term “gel” refersto a semi-solid formulation comprising a polymer network that is able totrap and contain fluids. As used herein, the term “semi-solid” refers tothe rheological properties of a formulation, such that the formulationwill flow under an applied force but will remain in situ followingapplication to any accessible body surface of a patient.

The chemical name of Nu-2 is((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((hydroxy(4-hydroxybutoxy)phosphoryl)oxy)-4-methoxytetrahydrofuran-2-yl)methyl(4-hydroxybutyl) hydrogen phosphate. The molecular formula of Nu-2 isC₁₈H₃₂N₂O₁₄P₂. Nu-2 has the following formula:

The chemical name of Nu-3 is(2R,3S)-2-((butoxy(hydroxy)phosphoryl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)butyl hydrogen phosphate. The molecular formula of Nu-3 isC₁₈H₃₂N₂O₁₁P₂. Nu-3 has the following formula:

The chemical name of Nu-4 is((2R,3S)-3-((butoxy(hydroxy)phosphoryl)oxy)tetrahydrofuran-2-yl) methylbutyl hydrogen phosphate. The molecular formula of Nu-4 is C₁₃H₂₈O₉P₂.Nu-4 has the following formula:

The chemical name of Nu-5 is Dibutyl (oxybis(ethane-2,1-diyl))bis(hydrogen phosphate). The molecular formula of Nu-5 is C₁₂H₂₈O₉P₂.Nu-5 has the following formula:

The chemical name of Nu-8 is((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-((butoxyoxidophosphor-yl)oxy)tetrahydrofuran-2-yl)methylbutyl phosphate. The molecular formula of Nu-8 is C₁₇H₂₉N₃Na₂O₁₀P₂. Nu-8has the following formula:

As used herein, the term “pharmaceutically acceptable,” with respect tosalts and formulation components such as carriers, excipients, anddiluents, refers to those salts and components which are not deleteriousto a patient and which are compatible with other ingredients, activeingredients, salts or components. Pharmaceutically acceptable includes“veterinarily acceptable,” and thus includes both human and non-humanmammal applications independently.

As used herein, the term “pharmaceutically acceptable salt” refers tosalts commonly used to form alkali metal salts and to form additionsalts of free acids or free bases. Such salts include, for example, thephysiologically acceptable salts listed in Handbook of PharmaceuticalSalts: Properties, Selection and Use, P. H. Stahl and C. G. Wermuth(Eds.), Wiley-VCH, New York, 2002, which are known to the skilledartisan. Salt formation can occur at one or more positions having labileprotons. The pharmaceutically acceptable salts of a Bisphosphocininclude both acid addition salts and base addition salts.

A Bisphosphocin is useful as an antimicrobial having activity againstany microbe. As used herein, the terms “microbe,” “microbial,” and liketerms refers to bacteria, fungi, protozoa, viruses, yeast, and the like.As used herein, the term “antimicrobial” refers to a Bisphosphocinhaving the ability to kill or inhibit the growth of a microbe, or toattenuate the severity of a microbial infection.

A non-limiting list of the bacteria that a Bisphosphocin is effectiveagainst include without limitation gram positive bacteria, gram negativebacteria, slow growing bacteria and acid fast bacteria, and any speciesincluded in the following genera: Aerococcus, Listeria, Streptomyces,Chlamydia, Lactobacillus, Eubacterium, Burkholderia, Stenotrophomonas,Achromobacter, Arachnid, Mycobacterium, Peptostreptococcus,Staphylococcus, Corynebacterium, Erysipelothrix, Dermatophilus,Rhodococcus, Pseudomonas, Streptococcus, Bacillus, Peptococcus,Pneumococcus, Micrococcus, Neisseria, Klebsiella, Kurthia, Nocardia,Serratia, Rothia, Escherichia, Propionibacterium, Actinomyces,Helicobacter, Enterococcus, Shigella, Vibrio, Clostridium, Salmonella,Yersinia, and Haemophilus.

Bisphosphocins can be used to treat nosocomial infections. Anon-limiting list of specific bacteria that cause nosocomial infectionsthat a Bisphosphocin is effective against include without limitationAcinetobacter iwoffii (clinical isolate), Acinetobacter baumannii(clinical isolate), Clostridium difficile (multi-resistant),Enterococcus faecalis (W.T. & vancomycin resistant), Enterococcusfaecium (vancomycin resistant), Klebsiella pneumoniae (clinical isolateand NDM-1), Pseudomonas aeruginosa (W.T.), Pseudomonas aeruginosa(ciprofloxacin, MDR), Serratia marcescens (oxacilllin resistant),Staphylococcus aureus (vancomycin), and Staphylococcus epidermis(oxacillin resistant).

Bisphosphocins can be used to treat community acquired infections. Anon-limiting list of specific bacteria that cause community acquiredinfections that a Bisphosphocin is effective against include withoutlimitation Aeromonas hydrophila (clinical isolate), Alcaligenes faecalis(clinical isolate), Borrelia burgdorferi, Haemophilus influenza,Mycobacterium tuberculosis (WT, MDR), Moraxella catarrhalis, Neisseriameningitidis (rifampicin resistant), Propionibacterium acnes, Proteusmirabilis, and Streptococcus pneumoniae (penicillin resistant).

Bisphosphocins can be used to treat food borne pathogens. A non-limitinglist of specific food borne pathogens that a Bisphosphocin is effectiveagainst include without limitation Escherichia coli (ampicillinresistant, NDM-1), Salmonella choleraesuis (enterica), and Salmonellatyphimurium (streptomycin resistant).

A non-limiting list of the fungi that a Bisphosphocin is effectiveagainst include without limitation Trichophyton, Epidermophyton,Microsporum, Candida albicans and other Candida species, Pityrosporumorbiculare, Trichophyton mentagrophytes, Trichophyton rubrum,Epidermophyton floccosurn, and Trichophyton tonsurans. A non-limitinglist of the viruses that a Bisphosphocin is effective against includewithout limitation human immunodeficiency virus (HIV), herpes simplexvirus (HSV), cytomegalovirus (CMV), Hepatitis B virus (HBV), Hepatitis Cvirus (HCV), and influenza virus. Unless specified to the contrary,specification of a compound of the present disclosure herein includespharmaceutically acceptable salts of such compounds.

A non-limiting list of specific fungal pathogens that a Bisphosphocin iseffective against include without limitation Trichophyton rubrum andmentagrophytes, Microsporum gypseum, and Aspergillus fumigatus.

In some embodiments, a Bisphosphocin is useful in both therapeutic andnon-therapeutic medical applications. In some embodiments includingnon-therapeutic medical applications, the antimicrobial effect of aBisphosphocin allows use of a Bisphosphocin for sterilization (e.g.,sterilization of a patient's skin or of a surface or an object, such as,for example, a surgical instrument), or sanitization (e.g., thecleansing of a surface, instrument, as to render the surface free ofundesirable concentrations of disease causing microorganisms). In someembodiments, the compounds of the present disclosure are effective incombating microbial contamination of laboratory cultures, consumables(e.g., food or beverage preparations), medical devices, hospitalapparatus, or industrial processes. Therapeutic applications of aBisphosphocin are described herein.

In some embodiments of the present disclosure, the gel formulationincludes a fatty alcohol thickening agent. As used herein, the term“fatty alcohol thickening agent” refers to a fatty alcohol compound orcomposition that increases the viscosity of a formulation of the presentdisclosure without substantially modifying such formulation's otherproperties. In some embodiments, the fatty alcohol thickening agent isselected from the group consisting of cetyl alcohol, lauryl alcohol,stearyl alcohol, cetosteryl alcohol, myristyl alcohol, docosanolalcohol, and oleyl alcohol, or combinations thereof. In someembodiments, the fatty alcohol thickening agent is cetosteryl alcohol.In some embodiments, the cetosteryl alcohol is present in theformulation in an amount from about 2% to about 10% (w/w). In someembodiments, cetosteryl alcohol is used such that the end viscosity isfrom 10 to 50000 centipoise (cps). In some embodiments, the endviscosity is from 500 to 20000 cps.

A particular challenge overcome by the formulations described herein isidentifying emulsifiers suitable for a low pH gel formation containingcharged molecules such as the Bisphosphocins. Typical ionic polymers,such as polyethylene glycol (PEG) esters, could not be used in thedisclosed gel formulations due to the electrostatic interactions withthe Bisphosphocins, leading the gel to collapse. Instead, anionicpolymers that could withstand low pH are needed.

In some embodiments of the present disclosure, the gel formulationincludes a nonionic polymer emulsifier. As used herein, the term“nonionic polymer emulsifier” refers to a nonionic surfactant. In someembodiments, the nonionic polymer is selected from the group consistingof polysorbate 20, polysorbate 60, polysorbate 80, ceteareth-12,ceteareth-20 (or Cetomacrogol 1000), ceteareth 30, ceteth-10, ceteth-20,steareth-10, steareth-20, steareth-40, and steareth-100. In someembodiments, the nonionic polymer emulsifier is selected from the groupconsisting of ester based products such as PEG-100 stearate, PEG-40stearate, PEG-120 glyceryl laurate, PEG-40 castor oil, PEG-60hydrogenated castor oil, PEG-75 lanolin, PEG-120 and methyl glucosedioleate. As used herein, “Cetomacrogol 1000” also means polyethyleneglycol hexadecyl ether.

In some embodiments, the nonionic polymer emulsifier is ceteareth 20. Insome embodiments, the ceteareth-20 is present in the formulation in anamount greater than about 0.5% (w/w). In some embodiments, theceteareth-20 is present in the formulation in an amount of about 0.5% toabout 5% (w/w).

In some embodiments, the formulation may include other pharmaceuticallyacceptable components to provide an improved formulation of aBisphosphocin, including without limitation other pharmaceuticallyacceptable carriers, excipients or diluents. The other carrier,excipient or diluent may take a wide variety of forms depending on theform of preparation desired for administration.

In some embodiments, the formulation further comprises a diluentselected from the group consisting of water, glycerol, mannitol, saline,and phosphate buffered saline. In some embodiments, the diluent iswater. In some embodiments, the water is present in the formulation inan amount from about 65% to about 97.5% (weight/weight).

In some embodiments of the present disclosure, the amount of theBisphosphocin present in the formulation varies. In some embodiments,the Bisphosphocin is present in the formulation in an amount from about1% to about 20% (weight/weight). In some embodiments, the Bisphosphocinis present in the formulation in an amount from about 5% to about 15%(weight/weight). In some embodiments, the Bisphosphocin is present inthe formulation in an amount from about 30% to about 50%(weight/weight).

In some embodiments of the present disclosure, the amount of the fattyalcohol thickening agent present in the formulation varies. In someembodiments, the fatty alcohol thickening agent is present in theformulation in an amount from about 1% to about 50% (weight/weight). Insome embodiments, the fatty alcohol thickening agent is present in theformulation in an amount from about 1% to about 20% (weight/weight). Insome embodiments, the fatty alcohol thickening agent is present in theformulation in an amount from about 1% to about 10% (weight/weight). Insome embodiments, the fatty alcohol thickening agent is present in theformulation in an amount from about 2% to about 8% (weight/weight).

In some embodiments of the present disclosure, the amount of thenonionic polymer emulsifier present in the formulation varies. In someembodiments, the nonionic polymer emulsifier is present in theformulation in an amount from about 0.25% to about 15% (weight/weight).In some embodiments, the nonionic polymer emulsifier is present in theformulation in an amount from about 0.5% to about 5% (weight/weight).

Because the Bisphosphocins as a class are more active at lower pH, it isnecessary to create suitable gel formulations that retain theantimicrobial activity of the Bisphosphocins while maintaining thephysical integrity of the gel. It is rare for gels to be formulated atpH levels in the desired pH range of about pH 1 to about pH 5, and evenmore so at pH 3 or below. The inventors of the present disclosuresuccessfully formulated a unique combination of the active ingredient,thickening agent, and emulsifier to achieve this goal. To achieve thedesired pH, in some embodiments, the formulation has a pH of about pH 1to about pH 5. In some embodiments, the formulation has a pH of about pH1.5 to about pH 4. In some embodiments, the formulation has a pH ofabout pH 3 to about pH 4.

The activity of the Bisphosphocins is pH dependent. Therefore, theformulations of some embodiments of the present disclosure also comprisea pH adjusting agent. In some embodiments, the pH adjusting agent can bean acid (such as 10% HCl), an acid salt, or mixtures thereof. Further,the pH adjusting agent can also be a buffer. Suitable buffers includecitrate/citric acid buffers, acetate/acetic acid buffers,phosphate/phosphoric acid buffers, formate/formic acid buffers,propionate/propionic acid buffers, lactate/lactic acid buffers,carbonate/carbonic acid buffers, ammonium/ammonia buffers, and the like.In some embodiments, the pH adjusting agent is present in an amountsufficient to adjust the pH of the composition to about pH 1.0 to aboutpH 5.0. In some embodiments, the pH adjusting agent is present in anamount sufficient to adjust the pH of the composition to about pH 2 toabout pH 4. In another aspect of the disclosure, the pH adjusting agentis present in an amount sufficient to adjust the pH of the compositionto about pH 3 to about pH 4.

In some embodiments, the formulations of the present disclosure areadapted for topical administration. As used herein, the term “topicaladministration” refers to administration of a Bisphosphocin to the skinsurface of a patient so that a Bisphosphocin passes through the skinlayer. Transdermal administration and transmucosal administration arealso encompassed within the term topical administration. As used herein,the term “transdermal” refers to passage of a Bisphosphocin across atleast one skin layer of a patient. As used herein, “transmucosal” refersto passage of Bisphosphocin across a mucous membrane of a patient.Unless otherwise stated or implied, the terms “topical administration,”“transdermal administration,” and “transmucal administration” are usedinterchangeably herein.

A variety of topical delivery systems for delivering bioactive compoundsto microbes in an patient are well known in the art. Such systemsinclude without limitation lotions, creams, gels, oils, ointments,solutions, suspensions, emulsions, and the like by choice of appropriatecarriers in the art.

In some embodiments, other materials may also be added to the topicalformulations of the present disclosure have additional moisturizingeffects and to improve the consistency of the formulation. Examples ofsuch compounds include without limitation cetyl esters wax, stearylalcohol, cetyl alcohol, glycerin, methyl paraben, propyl paraben,quaternium-15, humectants, volatile methylsiloxane fluids, andpolydiorganosiloxane-polyoxyalkylene. If it is desirable for theformulation to have additional cleaning effects in some embodiments,chemicals such as sodium lauryl sulfate or a metal salt of a carboxylicacid may be added.

The present disclosure also provides a method of treating an infectionin a patient in need thereof. In some embodiments, the method comprisesadministering an effective amount of a formulation of the presentdisclosure to the patient. As used herein, the term “infection” refersto any microbe infection of a patient's body. Infection includes theinvasion of a patient's body by a microbe and subsequent multiplicationin the patient's body.

As used herein, the terms “treating,” “treatment,” “therapy,” and liketerms refer to administration of a Bisphosphocin or formulation of thepresent disclosure in an amount effective to prevent, alleviate orameliorate one or more symptoms of a disease or condition (i.e.,indication) and/or to prolong the survival of the patient being treated.In some embodiments, “treating,” “treatment,” “therapy,” and like termsalso include without limitation reducing or eliminating infection in apatient.

In carrying out the methods of the present disclosure, an effectiveamount of a Bisphosphocin is administered to a patient in need thereof.As used herein, the term “effective amount,” in the context ofadministration, refers to the amount of a Bisphosphocin of the presentdisclosure that when administered to a patient is sufficient to prevent,alleviate or ameliorate one or more symptoms of a disease or condition(i.e., indication) and/or to prolong the survival of the patient beingtreated. Such an amount should result in no or few adverse events in thetreated patient. Similarly, such an amount should result in no or fewtoxic effects in the treated patient. As those familiar with the artwill understand, the amount of a Bisphosphocin will vary depending upona number of factors, including without limitation the activity of aBisphosphocin (in vitro, e.g. a Bisphosphocin vs. target, or in vivoactivity in animal efficacy models), pharmacokinetic results in animalmodels (e.g., biological half-life or bioavailability), the type ofpatient being treated, the patient's age, size, weight, and generalphysical condition, the disorder associated with the patient, and thedosing regimen being employed in the treatment.

In some embodiments, a Bisphosphocin is suitable for administration at adose of from about 1% to about 15%. In some embodiments, a Bisphosphocinis suitable for administration at a dose of from about 2% to about 10%.In some embodiments, a Bisphosphocin is suitable for administration at adose of from about 3% to about 8%. In some embodiments, a Bisphosphocinis suitable for administration at a dose of about 5%.

The present disclosure also provides a method of treating an infectionof a lower extremity ulcer in a patient in need thereof. In someembodiments, the method comprises administering an effective amount of aBisphosphocin, or a pharmaceutically acceptable salt thereof, to thepatient. As used herein, the term “lower extremity” refers to a lowerlimb of a patient's body, including without limitation the hip, thigh,leg, ankle, and foot. As used herein, the term “ulcer” refers to an openwound found anywhere on the lower extremity of a patient.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen. As used herein, the term “multiple dose regimen” refers to atreatment time period of more than one day.

In some embodiments, the present disclosure provides a method oftreating an infection of a diabetic foot ulcer in a patient in needthereof. In some embodiments, the method comprises administering aneffective amount of a Bisphosphocin, or a pharmaceutically acceptablesalt thereof, to the patient. In some embodiments, the patient issuffering from Type I diabetes or Type II diabetes. As used herein, theterm “diabetic foot ulcer” refers to an open wound located anywhere onthe foot of a patient. In some embodiments, the wound is located on theheel, mid-foot, and/or forefoot of the patient's foot. As used herein,the term “treating,” in the context of a diabetic foot ulcer, alsoincludes without limitation (a) limiting the progression in size, area,and/or depth of the foot ulcer; (b) reducing size, area, and/or depth ofthe foot ulcer; (c) increasing rate of healing and/or reducing time tohealing; (d) healing of the foot ulcer (100% epithelialization with nodrainage); and (e) decreased incidence of amputation or slowing in timeto amputation.

In some embodiments, the foot ulcer may be caused by any underlyingpathology, including but not limited to neuropathy, trauma, deformity,high plantar pressures, callus formation, edema, and peripheral arterialdisease. In some embodiments, the human diabetic foot ulcer is onecaused, at least in part, by neuropathy and resulting pressure (weightbearing on the extremity due to lack of feeling in the foot). As isknown to those of skill in the art, human diabetic foot ulcers tend tobe due to neuropathy and pressure, which differs significantly from, forexample, murine acute wounds. In some embodiments, the diabetic footulcer comprises one or more calluses.

In some embodiments, the diabetic foot ulcer is a chronic ulcer. As usedherein, a “chronic” foot ulcer is one that has been present for at least7 days with no reduction in size. In some embodiments, the chronic footulcer is one that has been present for at least 14 days. In someembodiments, the chronic foot ulcer is one that has been at least 21 or28 days with no reduction in size. In some embodiments, the chronic footulcer has not responded (i.e., no reduction in size, area, and/or depthof the foot ulcer; no healing of the foot ulcer) to any other treatment.

The methods of this disclosure can comprise administering the gelformulations described herein as often as deemed appropriate, i.e., onceper day, twice per day, etc. The methods according to the presentdisclosure may further comprise topical administration of theformulation containing a compound of the present disclosure, or apharmaceutically acceptable salt thereof, for as long as deemeddesirable by an attending physician, for example, until healing of theulcer. In some embodiments, the gel formulation forms a continuous filmcovering the entire area of the ulcer, including the margins. In someembodiments, the gel formulation is applied with a thickness ofapproximately 0.25 to 2 mm. In some embodiments, the gel formulation isapplied with a thickness of approximately 0.5 to 1.5 mm. In someembodiments, the topical formulation is applied with a thickness ofabout 1 mm in thickness.

The gel formulations disclosed herein may be applied in any suitablemanner, which may include any wound dressings to seal in the formulationdeemed appropriate by the human patient or caregiver. Examples of suchdressings include, but are not limited to, semipermeable films, foams,hydrocolloids, and calcium alginate swabs.

The methods of the present disclosure relating to diabetic foot ulcersmay further comprise debridement in and around the wound in combinationwith administration of the compound of the present disclosure andpharmaceutical compositions thereof. Debridement of all necrotic,callus, and fibrous tissue is typically carried for treatment ofdiabetic foot ulcers. Unhealthy tissue is sharply debrided back tobleeding tissue to allow full visualization of the extent of the ulcerand to detect underlying abscesses or sinuses. Any suitable debridementtechnique can be used, as determined by an attending physician. Thewound can then be thoroughly flushed with sterile saline or anon-cytotoxic cleanser following debridement.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

The present disclosure also provides a method of treating an infectionin a burn wound in a patient in need thereof. In some embodiments, themethod comprises administering an effective amount of a Bisphosphocin,or a pharmaceutically acceptable salt thereof, to the patient. As usedherein, the term “burn wound” refers to a burn injury to a patient'sbody involving damage to a patient's skin and possibly tissuesunderlying the patient's skin. There are three primary types of burnlevels known to one of skill in the art, including without limitationfirst, second, and third degree burns. In some embodiments, the methodof treating an infection in a burn wound contemplated by the presentdisclosure is used to treat a first, second, and/or third degree burn.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

In some embodiments, the present disclosure provides a method oftreating an infection of a complicated venous leg ulcer in a patient inneed thereof. In some embodiments, the method comprises administering aneffective amount of a Bisphosphocin, or a pharmaceutically acceptablesalt thereof, to the patient. As used herein, the term “complicatedvenous leg ulcer” refers to an open wound located anywhere on the leg ofa patient and resulting from improper functioning of veins.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

The present disclosure also provides a method of treating otitis externain a patient in need thereof. In some embodiments, the method comprisesadministering an effective amount of a Bisphosphocin, or apharmaceutically acceptable salt thereof, to the patient. As usedherein, the term “otitis externa” refers to an infection of the externalear canal of a patient.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

The present disclosure also provides a method of treating acne vulgarisin a patient in need thereof. In some embodiments, the method comprisesadministering an effective amount of a Bisphosphocin, or apharmaceutically acceptable salt thereof, to the patient. As usedherein, the term “acne vulgaris” refers to an inflammatory disease ofthe pilosebaceous glands characterized by an eruption of the skin, oftenpustular in nature but not suppurative.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

The present disclosure also provides a method of treating onychomycosisin a patient in need thereof. In some embodiments, the method comprisesadministering an effective amount of a Bisphosphocin, or apharmaceutically acceptable salt thereof, to the patient. As usedherein, the term “onychomycosis” refers to a fungal infection of thenail.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

The present disclosure also provides a method of treating conjunctivitisin a patient in need thereof. In some embodiments, the method comprisesadministering an effective amount of a Bisphosphocin, or apharmaceutically acceptable salt thereof, to the patient. As usedherein, the term “conjunctivitis” refers to inflammation or infection ofthe outer membrane of the eyeball and/or the inner eyelid.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

The present disclosure also provides a method of treating oral mucositisin a patient in need thereof. In some embodiments, the method comprisesadministering an effective amount of a Bisphosphocin, or apharmaceutically acceptable salt thereof, to the patient. As usedherein, the term “oral mucositis” refers to inflammation and ulcerationof the mucous membranes lining the mouth.

In some embodiments, the patient is a human. In some embodiments, theadministration is topical administration. In some embodiments, theadministration is carried out using a Bisphosphocin, or apharmaceutically acceptable salt thereof, in a lotion, paste, cream,ointment, oil or other viscous composition. In some embodiments, thepatient is administered at least one additional active ingredient. Insome embodiments, the administration is carried out as a multiple doseregimen.

The present disclosure also provides the use of a Bisphosphocin, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament. As used herein, the term “medicament” refers to aformulation according to the present disclosure. In some embodiments,the formulation is contained in any manufacture, such as, for example, apackage, container, and the like.

In some embodiments, Bisphosphocins that are useful in the formulationsaccording to the present disclosure have the structure of Formula (I),(II), (III), (IV), (V) or (VI):

wherein A is H, alkyl, alkyl-(O-alkyl), aryl, alkenyl, alkanol, phenol,or enol;

wherein Q is O, S, P—H, P—OH, P-alkyl, P-aryl, P-acyl, N—H, N—OH,N-alkyl, N-aryl, N-acyl, —CH₂, —CH(OH), or —CH(O-alkyl);

wherein X and Z are alkyl or O-alkyl end blocking groups; and

wherein W is H, purine, pyrimidine or a modified analog of a purine orpyrimidine; or

a pharmaceutically acceptable salt thereof. In some embodiments, Xand/or Z comprises an alkyl end blocking group, and wherein the alkylmoiety is straight chained, branched or cyclic. In some embodiments, thealkyl moiety has one to four carbons and is straight chained. In someembodiments, X and Z have the same chemical moiety. In some embodiments,X and Z have different chemical moieties. In some embodiments, X and Zcomprise a butyl group. In some embodiments, X is a butyl group and Z isa butanol. In some embodiments, X is a butanol and Z is a butyl group.In some embodiments, X and/or Z comprises a structure selected from thegroup consisting of CH₃CH₂CH₂CH₂—, CH₃CH₂CH₂—, CH₃CH₂—, andHOCH₂CH₂CH₂CH₂—.

In another embodiment, the compound has the Formula (VII):

wherein Ring A is a stable 5- to 7-membered monocyclic or 7-10-memberedbicyclic carbocyclic or heterocyclic moiety that is either saturated orpartially unsaturated on which a free amino group is attached to a ringcarbon atom, and R₁ and R₂ are each independently selected from thegroup of C₁-C₈ alkyl moieties, which may be branched or unbranched; or apharmaceutically acceptable salt thereof.

In some embodiments, R₁ and R₂ are both butyl:

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present disclosure, the Bisphosphocin for usein the formulations of the present disclosure include those having thestructure of Formula (IX) shown below:

or a pharmaceutically acceptable salt thereof.

All pharmaceutically acceptable salt forms of the compounds of Formulas(I)-(IX) are contemplated herein.

Another aspect of the present disclosure provides a method of treatingdiabetic foot ulcers in a patient in need thereof using the formulationsof this disclosure. In some embodiments, the method comprisesadministering an effective amount of a compound of the presentdisclosure by applying a formulation according to this disclosure to apatient. In some embodiments, the patient is a human.

Another aspect of the present disclosure provides a method of treatingburn wounds in a patient in need thereof using the formulations of thisdisclosure. In some embodiments, the method comprises administering aneffective amount of a compound of the present disclosure by applying aformulation according to this disclosure to the patient. In someembodiments, the patient is a human.

It is another advantage of an embodiment of the present disclosure thatthe formulation in accordance with some embodiments may be administeredin an effective amount to treat a patient suffering from adermatological disorder (such as, for example, complicated skin and skinstructure infections (cSSSI), acne vulgaris, otitis externa, oronychomycosis), an ophthalmic condition (such as, for example,conjunctivitis (pink eye), or a wound (for example, burn wounds,complicated diabetic foot ulcers (cDFCU) or complicated venous legulcers (cVLU)). In another embodiment, the gel formulation of thisdisclosure can be used during or after surgery (for example, inconnection with surgical incisions or implants).

The present disclosure also provides a method of treating venous legulcers in a patient in need thereof using the formulations of thepresent disclosure. In some embodiments, the method comprisesadministering an effective amount of a compound of the presentdisclosure by applying a formulation according to this disclosure to thepatient. In some embodiments, the patient is a human.

The present disclosure also provides a method of treating adermatological disorder in a patient in need thereof using theformulations of the present disclosure. In some embodiments, the methodcomprises administering an effective amount of a compound of the presentdisclosure by applying a formulation according to this disclosure to thepatient. In some embodiments, the patient is a human.

The present disclosure also provides a method of treating complicatedskin and skin structure infections in a patient in need thereof usingthe formulations of the present disclosure. In some embodiments, themethod comprises administering an effective amount of a compound of thepresent disclosure by applying a formulation according to thisdisclosure to the patient. In some embodiments, the patient is a human.

The present disclosure also provides a method of treating acne vulgarisusing the formulations of the present disclosure. In some embodiments,the method comprises administering an effective amount of a compound ofthe present disclosure by applying a formulation according to thisdisclosure to the patient. In some embodiments, the patient is a human.

The present disclosure also provides a method of treating otitis externain a patient in need thereof using the formulations of the presentdisclosure. In some embodiments, the method comprises administering aneffective amount of a compound of the present disclosure by applying aformulation according to this disclosure to the patient. In someembodiments, the patient is a human.

The present disclosure also provides a method of treating onychomycosisin a patient in need thereof using the formulations of the presentdisclosure. In some embodiments, the method comprises administering aneffective amount of a compound of the present disclosure by applying aformulation according to this disclosure to the patient. In someembodiments, the patient is a human.

The present disclosure also provides a method of treating an ophthalmiccondition in a patient in need thereof using the formulations of thepresent disclosure. In some embodiments, the method comprisesadministering an effective amount of a compound of the presentdisclosure by applying a formulation according to this disclosure to thepatient. In some embodiments, the patient is a human.

The present disclosure also provides methods for inhibiting orpreventing the growth of bacteria, fungus, or virus, by contacting theinfectious organism with a formulation disclosed herein. In someembodiments, the methods are used to treat a mammal. In certainembodiments, the methods are used to treat a human. In some embodiments,the present disclosure provides methods using a formulation of thisdisclosure to treat a mammal suffering from a bacterial infection causedby Staphylococcus aureus, Streptococcus pyogenes, Streptococcuspneumoniae, Mycobacterium, Escherichia coli, Aerococcus, Listeria,Streptomyces, Chlamydia, Lactobacillus, Eubacterium, Arachnid,Mycobacterium, Peptostreptococcus, Corynebacterium, Erysipelothrix,Dermatophilus, Rhodococcus, Pseudomonas (aeruginosa), Streptococcus,Bacillus, Peptococcus, Pneumococcus, Micrococcus, Neisseria, Klebsiella(pneumoniae), Kurthia, Nocardia, Serratia, Rothia, Escherichia,Propionibacterium, Actinomyces, Helicobacter, Enterococcus (Enterococcispp), Shigella, Vibrio, Clostridium, Salmonella, Yersinia, HaemophilusMorganella morganii, Proteus mirabilis, d coagulase-negativestaphylococci, coagulase negative Staphylococcus, Enterobacteriaceae, E.faecalis, and Acinetobacter baumannii

A range of fungi or molds, called dermatophytes, cause fungal infectionsof the skin. These fungi are parasites on the skin and cause differentsymptoms in different parts of the body. They are very infectious andare passed from person to person. Although typically these infectionsare topical, in certain patients (e.g., immunosuppressed patients) theymay occur systemically or internally.

Fungal infections that may be treated with the gel compositions of thepresent disclosure include dermatophytosis (Trichophyton,Epidermophyton, and Microsporum), candidiasis (Candida albicans andother Candida species), tinea versicolor (Pityrosporum orbiculare),tinea pedea (Trichophyton mentagrophytes, Trichophyton rubrum, andEpidermophyton floccosurn), tinea capitis and ringworm (Trichophytontonsurans).

Vaginal yeast infections are generally caused by Candida albicans,which, along with a few types of bacteria, are normally present inrelatively small numbers in the vaginal area. Sometimes the yeastmultiply rapidly and take over, causing candidiasis or monilia. This isoften due to a change in the vaginal environment, injury, sexualtransmission, HIV infection, etc. Common environmental disruptions thatfavor yeast include increased pH, increased heat and moisture, allergicreactions, elevated sugar levels, hormonal fluxes, and reductions in thepopulations of bacteria that are normally present.

In some embodiments, one dosage unit may be administered topically onceevery 10, 9, 8, 7, 6, 5, 4, 3, 2 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more than 10 times per day until relief is achieved or until thesymptoms disappear or are satisfactorily attenuated. In someembodiments, one dosage unit is administered topically about once toabout four times per day. In some embodiments, a patient is instructedto take two to three dosage units per day. Treatment regimens using thecompositions of the present disclosure can be acute or chronic. In someembodiments, particular dosing regimens include 7, 14, 21 and 28 daydosing.

The present disclosure also provides formulations and therapeuticmethods of using a compound of the present disclosure as an activeingredient in a gel formulation containing another active ingredient,e.g., an antibiotic, antifungal, an antiprotozoal, or antiviral. In someembodiments, a compound of the present disclosure is the only activeingredient administered to a patient to treat a microbial infection(such as a bacterial, fungal, viral or protozoan infection), i.e., acompound of the present disclosure is administered as a monotherapy. Themonotherapy may be administered with or without a treatment that is notspecific to an infection, such as a painkiller (e.g., acetaminophen or anonsteroidal anti-inflammatory drug such as aspirin, ibuprofen, ornaproxen) or numbing agent.

The present disclosure further provides disinfectant compositionscomprised of the formulations disclosed herein. The disinfectantcomposition may be suitable for use on skin, or may be used fordisinfection of a surface such as medical devices, e.g. a surgicalinstrument. The disinfectant composition may also be used in bandages orother dressings to prevent or treat wound infections.

The compounds disclosed for use in the formulations described herein maybe present in the form of pharmaceutically acceptable salts. Saltformation can occur at one or more positions having labile protons. Thepharmaceutically acceptable salts of the disclosed compounds includeboth acid addition salts and base addition salts. The term“pharmaceutically acceptable salts” embraces salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. Such salts include the physiologically acceptable salts listed inHANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, P. H.Stahl and C. G. Wermuth (Eds.), Wiley-VCH, New York, 2002 which areknown to the skilled artisan.

Suitable pharmaceutically acceptable acid addition salts of thedisclosed compounds may be prepared from an inorganic acid or an organicacid. Examples of such inorganic acids are hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which are formic, acetic, propionic, succinic,glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, beta-hydroxybutyric, malonic, galactic, and galacturonic acid,to name a few. Pharmaceutically acceptable acidic/anionic salts alsoinclude, the acetate, benzenesulfonate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate,iodide, isethionate, lactate, lactobionate, malate, maleate, malonate,mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, hydrogensulfate, tannate,tartrate, teoclate, tosylate, and triethiodide salts, among others.

Suitable pharmaceutically acceptable base addition salts of thedisclosed compounds include, but are not limited to, metallic salts madefrom aluminum, calcium, lithium, magnesium, potassium, sodium and zincor organic salts made from N,N′-dibenzylethylene-diamine,chloroprocaine, choline, diethanolamine, ethylenediamine,N-methylglucamine, lysine, arginine and procaine. All of these salts maybe prepared by conventional means from the corresponding compoundrepresented by the disclosed compound by treating, for example, thedisclosed compounds with the appropriate acid or base. Pharmaceuticallyacceptable basic/cationic salts also include, the diethanolamine,ammonium, ethanolamine, piperazine and triethanolamine salts, to name afew.

In an embodiment, the pharmaceutically acceptable salt comprises amonovalent cation or a divalent cation. In another embodiment, thepharmaceutically acceptable salt is a bis phosphate diester bis sodiumsalt.

A compound of the present disclosure may be co-formulated in the gelformulations described herein or co-administered with other activepharmaceutical agents depending on the condition being treated.Co-administration can include simultaneous administration of the twoagents in the same dosage form, simultaneous administration in separatedosage forms, and separate administration. In various embodiments,co-administration with respect to a compound of the present disclosuremeans either at the same time and frequency, or more usually, atdifferent times and frequencies than the compound of the presentdisclosure, as part of a single treatment plan. Aspects of the presentdisclosure include the administration of a compound of the presentdisclosure before, after, and/or during the administration of anotherantimicrobial agent. An antimicrobial agent (e.g., an agent thatgenerally or specifically targets a microbe) other than a compound ofthe present disclosure may therefore be used, in combination with acompound of the present disclosure, but yet be administered at differenttimes, different dosages, and at a different frequency, than a compoundof the present disclosure.

Doses and Dosing Regimes.

The presently described gel formulations comprising a compound of thepresent disclosure may be formulated with a variety of activeingredients and a variety of physiological carrier molecules. A compoundof the present disclosure may optionally be complexed with moleculesthat enhance their ability to enter the target cells. Examples of suchmolecules include, but are not limited to, carbohydrates, polyamines,amino acids, peptides, lipids, and molecules vital to bacterial growth.

Administration of the gel formulations of the present disclosure mayintroduce a compound of the present disclosure to the patient in adiluted amount. In some embodiments, unit dosages suitable for thepresent disclosure for topical administration may be more than about 1,5, 10, 50, 100, or 500 mg/kg.

In some embodiments of the present disclosure, the gel formulationscontain from about 0.001 percent by weight of a compound of the presentdisclosure to about 40 percent by weight. In some embodiments, the gelformulations contain from about 0.5% by weight of a compound of thepresent disclosure to about 30% by weight. In other embodiments, the gelformulations contain from about 1% by weight of a compound of thepresent disclosure to about 20% by weight. In yet other embodiments, thegel formulations contain from about 5% by weight of a compound of thepresent disclosure to about 20% by weight.

In one embodiment, one dosage unit may be topically administered onceevery 10, 9, 8, 7, 6, 5, 4, 3, 2, or one day, or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more than 10 times per day until relief is achieved or untilthe symptoms disappear or are satisfactorily attenuated. In someembodiments, one dosage unit is topically administered about once toabout four times per day. In some embodiments, dosing regimens fortopical administration include 1, 7, 14, 21 and 28 day dosing.

Therapeutic Use of Gel Compositions Containing a Compound of the PresentDisclosure.

The presently described formulations are also contemplated to beeffective in combating microbial (e.g., bacterial, fungal, protozoan, orviral) contamination of laboratory cultures, consumables (food orbeverage preparations), medical devices, hospital apparatus, orindustrial processes.

The gel formulations disclosed herein are particularly useful fortreating infections caused by biofilms. Biofilms form when singlemicroorganisms attach to a hydrated surface and grow as an adhesive cellmatrix with other microorganisms. The biofilms form densely packedcommunities of microbial cells which surround themselves with secretedpolymers. Biofilms are notoriously difficult to treat and have beenimplicated in diseases as disparate as atherosclerosis, chronicsinusitis and chronic wound healing.

Bactericidal and/or bacteriostatic activity of the gel formulationsincluding a compound of the present disclosure, or a pharmaceuticallyacceptable salt thereof, may be measured using any number of methodsavailable to those skilled in the art. One example of such a method ismeasurement of antibacterial activity through use of a MIC (minimalinhibitory concentration) test that is recognized to be predictive of invivo efficacy for the topical treatment of a bacterial infection withantibiotics. The gel formulations of the present disclosure displayantibacterial activity in this test, even without pretreatment of thebacteria to permeabilize the membrane.

The present disclosure provides methods of inhibiting the growth ofmicroorganisms by contacting the microorganisms with the gelformulations of the present disclosure in which the active agent is acompound of the present disclosure. These methods are effective againstinfections in vivo, and particularly topical infections. This isdemonstrated by test data showing the minimum inhibitory concentrations(MIC) and minimum biocidal concentrations (MBC) of formulations againstvarious pathogenic organisms cultured in vitro under standardconditions. These in vitro tests strongly correlate with in vivoactivity, as is evidenced by the widespread use of the MIC and MBCdeterminations to predict utility of antimicrobial formulations intreatment of infection in animals, including humans.

Particularly striking is the ability of the present gel formulationscomprising a compound of the present disclosure to extend the range ofantimicrobial effectiveness against bacteria previously consideredunreactive towards certain conventional antibiotics. For example, a gelformulation comprising a compound of the present disclosure may beespecially useful in formulations to treat acne, diabetic foot ulcers,otitis externa, or burn wounds.

A compound of the present disclosure, as well as having, antibacterialactivity, may also have activity as an antifungal. A compound of thepresent disclosure is thus useful as an active agent in the gelformulation for treatment of fungal infections such as tinea pedea andcandidiasis.

Gel formulations comprising a compound of the present disclosure mayalso have activity as an antiviral. Such formulations are thus useful asa topical treatment for viral infections such as herpes simplex.

The gel formulations disclosed herein may also be used as topicaldisinfectants for sterilization of surfaces such as, for example,countertops, surgical instruments, bandages, and skin; as pharmaceuticalformulations, for external application to skin (e.g., as a handsanitizer) and mucosal surfaces (e.g., a nasal sanitizer), including thecornea, dermal cuts and abrasions, burns, and sites of bacterial orfungal infection; as pharmaceutical formulations, for administration tointernal mucosal surfaces such as the oral cavity or vagina to inhibitthe growth of bacteria, viruses or fungi, including yeasts; and aspharmaceutical formulations for coating indwelling catheters and similarimplants which are susceptible to harboring bacterial or fungalinfection.

Antibiotics.

The gel formulations of this disclosure may be useful as topicalantibiotic formulations including other antibiotic agents, with bothprescription agents (e.g., benzomycin) and over-the-counter agents(e.g., salicylic acid, benzoyl peroxide and the like). When used in thetherapeutic treatment of disease, an appropriate dosage of a gelformulation containing a compound of the present disclosure and anotheractive ingredient may be determined by any of several well establishedmethodologies. For instance, animal studies are commonly used todetermine the maximal tolerable dose, or MTD, of bioactive agent perkilogram weight. In general, at least one of the animal species testedis mammalian. Those skilled in the art regularly extrapolate doses forefficacy and avoiding toxicity to other species, including human.Additionally, therapeutic dosages may also be altered depending uponfactors such as the severity of infection, and the size or species ofthe host.

Antivirals.

Because some of the compounds of the present disclosure are based on thenatural structures of nucleosides, formulations comprising compounds ofthe present disclosure may possess efficient antiviral activity.

Viruses that may be treated by the formulations of the presentdisclosure include, but are not limited to, human immunodeficiency virus(HIV), herpes simplex virus (HSV), cytomegalovirus (CMV), Hepatitis Bvirus (HBV), Hepatitis C virus (HCV) and influenza virus.

Conjunctive Therapies.

The gel formulations comprising a compound of the present disclosure canalso be used in conjunction with conventional antimicrobial agents. Theadded activity of the active ingredients may provide for a moreefficacious formulation, and can provide multiple mechanisms by whichthe microbes are targeted. Pharmaceutical compositions containing acompound of the present disclosure in admixture with another activeagent can be prepared according to conventional pharmaceuticalcompounding techniques.

For example, compositions for the treatment of acne may comprise acompound of the present disclosure with salicylic acid, benzoylperoxide, and/or sulfur. Such conjunctive therapy using a compound ofthe present disclosure with another active agent can increase theefficacy of gel formulations of this disclosure without having toincrease the amounts of the agents currently available to consumers,e.g., the amount found in over-the-counter products.

Over the counter (OTC) antifungal medications that may be additionalactive ingredients in the gel compositions of the present disclosureinclude, but are not limited to: Miconazole, Miconazole nitrate,Polynoxylin, Clotrimazole, Sulconazole nitrate, Econazole nitrate,Tolnaftate, Selenium sulphide, Tioconazole Presciptive antifungalsinclude drugs such as allylamines, azoles, polyene macrolides,flucytosine, pseudomycins and griseofulvin. Exemplary antifungalsinclude Amphotericin B, Fluconazole/Difluian, Flucytosine, Foscarnet,Itraconazole/Sporonex, Ketoconazble/Nitoral, and Nystatin 1. See alsoElewski, Cutaneous Fungal Infections, 2nd Edition (1998) and Segal,Pathogenic Yeasts and Yeast Infections (1994), which are bothincorporated by reference.

The gel formulations of the present disclosure contain a compound of thepresent disclosure as an active ingredient, and may also contain any ofa number of additives that are themselves active ingredients, such as aretinoic acid, glycolic acid, lactic acid, α-hydroxy acids, keto-hydroxyacids, citric acid, glucuronic acid, galacturonic acid,glucuronolactone, gluconolactone, a-hydroxy-butyric acid,α-hydroxyisobutyric acid, malic acid, pyruvic acid, phenyllactic acid,phenylpyruvic acid, saccharic acid, mandelic acid, tartaric acid,tartronic acid, hydroxybutyric acid, vitamin A palmitate (retinylpalmitate) and/or vitamin E acetate (tocopheryl acetate). In someembodiments, each of these are present in an amount from about 0.5 wt %to about 20 wt %. In addition, a UV absorbing or blocking material, suchas 4-aminobenzoic acid (PABA), may be used.

Additional active ingredients that may be used in the gel formulationsof this disclosure include those found in U.S. Pat. No. 5,652,266,directed to combination of α-hydroxy acid, retinoid and salicylic acid;U.S. Pat. No. 5,843,998, directed to a composition containing α-hydroxyacids and carbamide peroxide, either with or without salicylic acid;U.S. Pat. No. 5,153,230, which is directed to a formulation in which themajor active ingredient is glycolic acid; U.S. Pat. No. 4,464,392, whichis directed to a antimicrobial formulations containing glycolic acidderivatives; and U.S. Pat. No. 4,105,782, which describes numerous othersimilar active agents that may be used in a gel composition of thepresent disclosure.

The gel formulations of the present disclosure may also be used as acarrier material for and/or in combination with other medicines, such asspermicidal agents, anti-viral agents and anti-fungal agents, therebyfurther broadening the compositions medical efficacy. The gelformulations of the present disclosure may also include a topicalanesthetic such as lidocaine hydrochloride and topical steroids, such ascorticosteroid, to provide relief from pain or itching during treatment.

Use of Gel Formulations as Disinfectants.

The gel formulations described herein containing compounds of thepresent disclosure may also find use as a disinfectant, and aspreparations having biostatic or biocidal properties. The disinfectantgel formulations of the present disclosure may also contain other activeingredients with biostatic and/or biocidal properties. For example, thedisinfectant gel may contain a compound of the present disclosure with asuitable concentration of a quaternary ammonium compound such as:dimethylbenzyldodecylammonium chloride, dimethylbenzyl decylammoniumchloride, dimethylbenzyl decylammonium bromide, anddimethylbenzylloctylammonium chloride.

In another example, suitable microbiocidal biguanidine compounds, suchas oligohexamethylene biguanide salts and bisbiguanides, can be used.See, e.g., U.S. Pat. No. 5,030,659. Additional biocidal ingredientsinclude aldehydes, phenol derivatives, and halogen phenyl derivatives.See, e.g., U.S. Pat. No. 5,767,054. Other compounds with such activity,as will be recognized by those skilled in the art, may also be used inconjunction with compounds of the present disclosure in the gelformulations described herein.

These preparations are especially suitable for surface disinfection inmedically-related environments, such as hospitals, veterinary clinics,dental and medical offices, and the like. In some embodiments,formulations of the present disclosure are used in the sterilization ofsurgical instruments. These preparations are also useful in public areassuch as schools, public transport, restaurants, hotels and laundries.The disinfectants also find use in home as sanitizers for toilets,basins, and kitchen areas.

EXAMPLES

Examples related to the present disclosure are described below. In mostcases, alternative techniques can be used. The examples are intended tobe illustrative and are not limiting or restrictive of the scope of theinvention as set forth in the claims.

The compounds of the present disclosure are synthesized according tomethods known to those of ordinary skill in the art. The methodsdescribed in U.S. Pat. Nos. 6,627,215, 6,211,162, 7,868,162, 7,176,191,8,435,960, and 6,211,349, all of which are hereby incorporated byreference in their entireties, are suited for synthesizing the compoundsof the present disclosure. Nu-8 is synthesized according to thefollowing method:

Step-1. Cytidine 2a is added to hexamethyldisilazane (HMDS),dimethylamino pyridine (DMAP) and di-tert-butyl dicarbonate ((BOC)₂O) toprotect the nitrogen atoms of 2a by generating BOC-protected compoundInt-2.

Step-2: n-Butanol is reacted with the phosphinamide 1 in THF withtetrazole as a catalyst in the presence of di-isopropylethylamine(DIPEA). The crude product is chromatographed on neutral alumina elutingwith hexane and then 2% ethyl acetate in hexane. The pure fractions iscombined (by TLC) and evaporated to a residue under vacuo.

Step-3. BOC-protected species Int-2 is bis-phosphinylated with reagent 2in dichloromethane (DCM)/dimethyl formamide (DMF) solvent and tetrazoleas catalyst to produce 3. The reaction mixture is concentrated to aresidue and the crude product is immediately oxidized in the next step.

Step-4 &5: The crude product 3 is oxidized with t-butylhydroperoxide(TBTH) in the presence of decane to generate the bis-phosphonate species4. Removal of the BOC groups is carried in DCM in the presence oftrifluoro acetic acid (TFA) to yield 5. The crude product ischromatographed on silica gel eluting with ethyl acetate. The purefractions (by TLC) are combined and evaporated to a residue under vacuo.

Step-6: Hydrolysis of 5 with methanolic ammonium hydroxide (NH₄OH, MeOH)gives crude (I) ammonium salt (6).

Step-7 &8: Purification by preparative HPLC of 6 and conversion to thefree acid with Dowex 50WX8-200 resin is carried out. Evaporation of theaqueous eluate provides (I) that is diluted with purified water toprovide a 20% solution at its ambient pH.

The sodium salt of the compound having the Formula (IX), or Nu-8, issynthesized according to the following method:

Synthesis of Compound-2: To a solution of compound-1 (1.0 kg, 3.3222mol) in THF (6 L) is added DIPEA (1.370 ml, 8.3055 mol) and Tetrazole(230 g, 3.3222 mol) followed by n-Butanol (275 ml, 2.99 mol) in THF (6L) is added drop wise at 0° C. for 12 h. The reaction mixture is stirredat room temperature for 24 h. The progress of the reaction is monitoredby TLC and after completion of the reaction, solid is filtered off.Filtrate is evaporated under reduced pressure at 40° C. to afford crudecompound. Crude compound is dissolved in ethyl acetate (5 L). Organiclayer is washed with water (3 L) and brine (2 L). Organic layer is driedover anhydrous Na₂SO₄ filtered and the solvent is evaporated underreduced pressure to get afford crude Compound. The Crude compound ispurified by column Chromatography over Basic Alumina (Al₂O₃), Compoundeluted with 0-2% EtOAc in pet ether to afford compound-2. (700 g,76.92%) as pale yellow liquid. H-NMR (400 MHz, Chloroform-d) δ 4.18-4.07(m, 1H), 4.02 (q, J=6.6 Hz, 1H), 3.93-3.74 (m, 4H), 2.65 (td, J=6.5, 3.6Hz, 2H), 1.31-1.23 (m, 4H), 1.18 (dd, J=6.8, 3.8 Hz, 12H), 0.93 (td,J=7.4, 3.1 Hz, 3H). LC-MS: 275 (M+H).

Synthesis of Compound-4: To solution of compound-3 (300 g, 1.321 mol) inHexamethyldisilazane (638 g, 3.964 mol) is added DMAP (16.11 g, 0.132mol) followed by TMSOTf (7.22 g, 0.039 mol) is added at 0° C. and theresulting reaction mixture is stirred for 1 h at room temperature. Aftercomplete of starting material Boc-anhydride (1.4 L, 6.605 mol) is addedat 0° C. for 1 h and the reaction mixture is stirred for 16 h at roomtemperature. To the reaction is added methanol (3 L) followed bytriethylamine (1.5 L) is added at 0° C. for 1 h and the reaction mixtureis stirred for 20 h at room temperature. Reaction mixture isconcentrated under reduced pressure to get crude compound. Crudecompound is diluted with ethyl acetate (3 L) and washed with water (1.0L) and brine (1.0 L) solution; Organic layer is dried over anhydrousNa₂SO₄, filtered and the solvent is evaporated under reduced pressure toget afford crude compound. The Crude compound is purified by columnChromatography silica gel (100-200 mesh) Compound eluted 0-3% MeOH inDCM to afford compound-4 (180 g, 31.89%) as off white solid. H-NMR (300MHz, DMSO-d6) δ 8.41 (d, J=7.5 Hz, 1H), 6.84 (d, J=7.5 Hz, 1H), 6.06 (t,J=6.2 Hz, 1H), 5.28 (d, J=4.3 Hz, 1H), 5.07 (q, J=4.6, 4.0 Hz, 1H), 4.21(q, J=4.1 Hz, 1H), 3.87 (q, J=3.7 Hz, 1H), 3.71-3.49 (m, 2H), 2.32 (m,1H), 2.03 (dt, J=13.0, 6.2 Hz, 1H), 1.49 (s, 18H). LC-MS: 275 (M+H).

Synthesis of Compound-6: To a stirred solution of compound-4 (180 g,0.421 mol) in THF (1.0 L) is added DIPEA (348 mL, 2.105 mol) andTetrazole (176 g, 2.526 mol) at 0° C. To the resulting reaction mixtureis added a solution of compound-2 (519 g, 1.896 mol) in THF (800 ml)drop wise at 0° C. for 1 h. The reaction mixture is stirred at roomtemperature for 16 h. After completion of the reaction, tert-butylperoxide in decane (505 ml, 5M) is added drop wise at 0° C. and thereaction mixture is stirred for 6 h at room temperature. The reaction ismonitored by TLC. After completion of the reaction, the reaction mixtureis concentrated at 40° C. and diluted with ethyl acetate (3 Lit) andwashed with water (1 Lit) and brine (1 Lit) solution; Organic layer isdried over anhydrous Na₂SO₄ filtered and the solvent is evaporated underreduced pressure to get afford crude compound (350 g, crude). The Crudecompound is purified by column chromatography through silica gel(100-200 mesh) column eluted with 0-5% MeOH in DCM. All collected purefractions are concentrated to afford pure compound-6 (220 g, 64.83%) asa wine red liquid. H-NMR (300 MHz, DMSO-d₆) δ 8.19 (dd, J=7.6, 1.3 Hz,1H), 6.88 (d, J=7.5 Hz, 1H), 6.13 (t, J=10.5 Hz, 1H), 4.99 (s, 1H), 4.44(s, 1H), 4.26-3.96 (m, 10H), 3.00-2.84 (m, 4H), 2.57-2.79 (m, 2H),1.70-1.54 (m, 4H), 1.50 (s, 18H), 1.35 (m, 4H), 0.88 (qd, J=7.5, 2.5 Hz,6H); LC-MS: 806 (M+H).

Synthesis of Compound-7: To a solution of compound-6 (220 g, 0.273 mol)in DCM (4.4 L) is added TFA (210 mL, 2.732 mol) drop wise at 0° C. Thereaction mixture is stirred at room temperature for 24 h. The reactionis monitored by TLC. After completion of the reaction, solvent isevaporated under reduced pressure to afford Crude compound. The Crudecompound is purified by column Chromatography silica gel (230-400 mesh)Compound eluted with 0-10% MeOH in DCM. All collected pure fractions areconcentrated to afford pure compound-7 (170 g, 84.67%) as a pale yellowliquid. H-NMR (300 MHz, DMSO-d₆) δ 7.61 (d, J=7.5 Hz, 1H), 7.27 (d,J=13.9 Hz, 2H), 6.19 (t, J=6.9 Hz, 1H), 5.74 (d, J=7.4 Hz, 1H), 4.96 (s,1H), 4.10-3.93 (m, 11H), 2.93 (q, J=6.2 Hz, 4H), 2.29 (d, J=13.1 Hz,2H), 1.61 (h, J=7.1 Hz, 4H), 1.35 (p, J=7.3 Hz, 4H), 0.89 (dq, J=7.9,4.2 Hz, 6H); LC-MS: 606 (M+H).

Synthesis of Nu-8: To a stirred solution of compound-7 (720 g, 1.1900mol) in MeOH (5.0 L) is added aq. ammonia (600 mL) at 0° C. The reactionmixture is stirred at room temperature for 4 h. The reaction ismonitored by TLC. After completion of the reaction, evaporate the MeOHunder reduced pressure the aqueous layer is washed with DCM (1.5 L). Theaqueous layer is passed through Dowex-H⁺ resin. The water is removedunder reduced pressure to afford Nu-8 (260 g, 43.84%) as an off whitesolid. H-NMR (300 MHz, DMSO-d₆) δ 8.94 (s, 1H), 8.49 (s, 1H), 7.97 (d,J=7.8 Hz, 1H), 6.08 (t, J=6.1 Hz, 1H), 5.95 (d, J=7.7 Hz, 1H), 4.76 (q,J=5.8 Hz, 1H), 4.15 (q, J=4.1 Hz, 1H), 4.08 (s, 1H), 3.83 (m, 6H), 2.43(t, J=5.6 Hz, 2H), 1.67-1.44 (m, 4H), 1.44-1.26 (m, 4H), 0.95-0.82 (m,6H). LC-MS: 500.15 (M+H).

Synthesis of Nu-8 Sodium salt: To a stirred solution of compound-Nu-8(260 g, 0.478 mol) in water (2.6 L), 1N NaOH (950 mL) is added drop wiseat 0° C. The reaction mixture is stirred at room temperature for 2 h.The reaction is monitored by TLC. After completion of the reaction,aqueous layer is washed with DCM (1.5 L). The aqueous layer isevaporated under reduced pressure to afford Nu-8 Sodium salt (265 g,93%) as off white solid. H-NMR (300 MHz, DMSO-d₆) δ 7.81 (d, J=7.2 Hz,1H), 7.2 (bs, 1H), 7.0 (bs, 1H), 6.16 (t, J=4 Hz, 1H), 5.71 (d, J=7.6Hz, 1H), 4.69 (bs, 1H), 3.75 (m, 1H), 3.71 (m, 1H), 3.8 (m, 4H), 2.2 (q,1H), 1.89-1.96-1.44 (m, 1H), 1.49-1.39 (m, 4H), 1.34-1.23 (m, 4H),0.88-0.84 (m, 6H).

All excipients referred to in the following Examples are compendialgrade. All solvents for analysis are HPLC grade.

HPLC analysis of Nu-3 is performed using HPLC. The HPLC instrument is anAgilent 1200 system with a diode array detector. Approximately 0.2 g ofa Nu-3 fatty alcohol-based gel is added to a 50 mL volumetric flask. Fora Nu-3 cellulose-based gel, ˜25 mL of water is added, the flask isvortexed to dissolve the gel, then the flask is brought to volume withwater. For the fatty alcohol (FA) gel, ˜25 mL of warm water (45-50□) isadded with a small stir bar, the flask is mixed for 15 minutes touniformly disperse the FA gel, the stir bar is removed and the flask isbrought to volume with water. The cellulose gel extract is suitable fordirect injection; an aliquot of the FA gel extract is centrifuged for 2minutes to remove undissolved solids.

An Orion 710 A+ meter and electrode (Thermo) are used to measure pH.Viscosity is measured with a Brookfield viscometer. Moderate shearmixing for gels are provided by an IKA Eurostar 200 overhead drive witha 1.0″ marine propeller. High shear mixing for FA gels is provided by aOmni homogenizer with a 7 mm rotor-stator head at ˜10,000 rpm.

Example 1: Cellulose Gel Compounding

The following steps are used to prepare cellulose gels using a solutionof Nu-3 as the free acid at the 100 g scale.

1. Add Nu-3 to a portion of the water and mix until homogeneous.

2. Add sodium chloride and mix until homogeneous.

3. Adjust pH to 1.5 (1.4-1.6 acceptable range) using 4% NaOH.

4. Add remaining water and mix until homogeneous.

5. Slowly add hydroxyethyl cellulose powder (Natrosol 250 HHX PH,Ashland) to the vortex of the mixing propeller.

6. Continue mixing until the polymer gel is transparent (˜45-60minutes).

Example 2: Fatty Alcohol (FA) Gel Compounding

The following steps are used to prepare FA gels using a solution of Nu-3as the disodium salt at the 100 g scale.

1. Add Nu-3 to a portion of the water and mix until homogeneous.

2. Adjust pH to 1.5 (1.4-1.6 acceptable range) using 10% HCl.

3. Add remaining water and mix until homogeneous.

4. In a separate vessel combine cetostearyl alcohol (Crodacol CS 50 NF,Croda) and ceteareth-20 (Cetomacrogol 1000 NF, Croda) and heat to ˜60°C. on a hot plate with mixing to melt the fatty alcohol and surfactant.Hold at ˜60° C.

5. Heat API solution to ˜60° C. on a hot plate while mixing with thepropeller mixer.

6. Add fatty alcohol/surfactant mixture to the API solution while mixingwith the propeller mixer. Remove propeller mixer, remove vessel fromheat, and start high shear mixing.

7. Continue high shear mixing as the gel cools and thickens (˜45-50°C.).

8. When the gel becomes too thick for mixing with the homogenizer, stophigh shear mixing and continue mixing with the propeller mixer until thegel reaches 35-40° C.).

Example 3: Set-Up HPLC Assay and Autoclaving Study

The chromatography conditions in Table 1 are used to assay Nu-3formulations for this Example.

TABLE 1 Chromatography conditions. Column Phenomenex Kinetex XB-C18 4.6× 250 mm, 3.5 μm Guard column Phenomenx SecurityGuard cartridge, C18(ODS) 4 mm L × 3 mm ID Mobile Phase A 10 mM ammonium acetate MobilePhase B Acetonitrile Gradient  0.0 min. 95% A 20.0 min.  5% A 25.0 min. 5% A 25.1 min. 95% A 30.0 min. 95% A Run Time 30 min. Flow Rate 1.0mL/min. UV Detector 265 nm Injection Volume 10 μL Column Temperature 30°C.

Linearity is evaluated using Nu-3 solutions from 0.05 to 0.4 mg/mL. Thecorrelation coefficient for peak area versus mg/mL has a value of0.9994. The % Relative Standard Deviation (RSD) for repeated injectionsof a 0.2 mg/mL standard is <1.0%.

Example 4: Vehicle Gels for Evaluation

Three vehicle gels are prepared for evaluation. Sodium phosphate is usedto simulate the presence of the compound of Formula (I) and benzylalcohol is used as an antimicrobial preservative. Their compositions aresummarized in Table 2.

TABLE 2 Vehicle gel compositions. All numbers are % w/w. Ingredient Gel1 Gel 2 Gel 3 Sodium phosphate monobasic 0.4 0.4 0.4 (monohydrate), USP3.5% HCl (q.s. to pH 2.5) 0.9 0.9 0.9 Sodium chloride, USP 0.32 0.320.32 Benzyl alcohol, NF 0.5 0.5 0.5 Natrosol HXX1250 NF 1.00 1.75 0.0Crodacol CS 50 NF 0.0 0.0 4.0 Cetomacrogol 1000 NF 0.0 0.0 1.0 Purifiedwater, USP q.s. to 100% q.s. to 100% q.s. to 100%

Gels 2 and 3 are selected for formulation with Nu-3 at 5% w/w due totheir superior physical properties. The formulation pH is reduced to atarget value of 1.5 to ensure optimal activity of Nu-3.

Example 5:5% Nu-3 Cellulose Gel: Formulation and Stability

The composition for this gel is shown in Table 3.

TABLE 3 5% cellulose gel composition. All numbers are % w/w. Ingredient5% Nu-3 cellulose gel Nu-3 20% solution 25.0 4% NaOH (q.s. to pH 1.5)3.0 Sodium chloride, USP 0.32 Natrosol HXX250 NF 1.75 Purified water,USP q.s. to 100%

The results for initial and stability results for the 5% Nu-3 cellulosegel are summarized in Table 4.

TABLE 4 Initial and stability results for the 5% Nu-3 cellulose gel.Time point/storage Assay, condition % w/w Viscosity, cP¹ Appearance pHInitial 4.94 12,200 Viscous gel 1.43 1 month, 40° C. 4.90 125 Liquid1.51 1 month, 30° C. Not tested 4,600 Liquid Not tested 1 month, 25° C.Not tested 6,870 Slightly viscous Not tested liquid 1 month, 5° C. Nottested 11,200 Viscous gel Not tested ¹RV viscometer, S14 spindle, 6Rsmall sample adaptor, 30 rpm.

On storage, the cellulose Nu-3 gel's viscosity decreases significantlywith temperature. This is likely due to hydrolysis of the cellulose inthe polymer. However, the assay and pH after 1 month of storage at 40°C. has no significant change.

Example 6:5% Nu-3 Fa Gel: Formulation and Stability

The composition for these gels are shown in Table 5.

TABLE 5 5% FA gel compositions. Ingredient Nu-3 FA Gel 1 Nu-3 FA Gel 2Nu-3 disodium salt 5.36 5.36 10% HCl (q.s. to pH 1.5) 2.8 2.8 CrodacolCS 50 NF 4.0 7.25 Cetomacrogol 1000 NF 1.0 1.0 Purified water, USP q.s.to 100% q.s. to 100%

During compounding, FA Gel 1 fails to thicken.

For FA Gel 2, the cetostearyl alcohol level is increased from 4.0 to7.25% w/w. This increases the gel viscosity for the vehicle and the 5%Nu-3 formulations. The stability data for Nu-3 FA Gel 2 are summarizedin Table 6.

TABLE 6 Initial and stability results for the 5% Nu-3 FA Gel 2. Timepoint/storage Assay, condition % w/w Viscosity, cP¹ Appearance pHInitial 4.90 60,000 Viscous, off white gel 1.52 1 month, 40° C. 4.9674,000 Viscous, off white gel 1.45 1 month, 25° C. 4.88 64,000 Viscous,off white gel 1.55 ¹RV viscometer, S14 spindle, 6R small sample adaptor,0.6 rpm.

The assay, appearance, and pH for FA Gel 2 shows no significant changeafter 1 month at 25 or 40° C. There is a slight increase in viscosity onstorage, which is not uncommon for fatty alcohol gels. Their viscositiestend to level off after 1-3 months of storage.

Higher Strength Nu-3 FA Gels: Formulation and Stability

The composition for these gels are shown in Table 7 and the stabilityresults are shown Table 8.

TABLE 7 FA gel compositions. All numbers are % w/w. 10% Nu-3 15% Nu-320% Nu-3 Ingredient FA Gel FA Gel FA Gel Nu-3 disodium 10.8 16.2 21.610% HCl (q.s. to pH 1.5) 5.0 6.6 7.8 Crodacol CS 50 NF¹ 7.25 7.25 7.25Cetomacrogol 1000 NF¹ 1.0 1.0 1.0 Purified water, USP q.s. to 100% q.s.to 100% q.s. to 100% ¹Substution of another vendor’s grade of thisexcipient may cause a significant change in the formulation and is notrecommend.

TABLE 8 Stability results for the Nu-3 FA Gels. Time point/ Assay,Viscosity, storage condition % w/w cP¹ Appearance² pH Initial 10% 10.363,500 Conforms 1.45 15% 14.6 65,200 Conforms 1.52 20% 19.9 71,000Conforms 1.59 1 month/40° C. 10% 10.2 85,600 Conforms 1.53 15% 14.789,000 Conforms 1.48 20% 20.1 84,400 Conforms 1.63 1 month/25° C. 10%10.1 66,900 Conforms 1.44 15% 14.6 69,000 Conforms 1.51 20% 19.8 72,400Conforms 1.55 ¹RV viscometer, S14 spindle, 6R small sample adaptor, 0.6rpm. ²Off-white to tan viscous gel

The assay, appearance, and pH for FA gels with 10-20% Nu-3 shows nosignificant change after 1 month at 25 or 40° C. There is a slightincrease in viscosity on storage, which is not uncommon for fattyalcohol gels. Their viscosities tend to level off after 1-3 months ofstorage.

Example 7: Evaluation of Efficacy of a Bisphosphocin in A Staphylococcusaureus-Induced Murine Dermal Infection Model Animals and Husbandry

Female SKH1 mice ordered from Charles River Laboratories are acclimatedto housing conditions and handled in accordance with Animal Use Protocol(AUP) number TP-18. The animals are acclimated for a minimum of 24 hoursprior to bacterial challenge and are 6-8 weeks old on Day 0 of theexperiment. Only animals deemed healthy are included in this study.Animals are fed irradiated Teklad Global Rodent Diet 2918 and water adlibitum. Mice are housed in static cages with irradiated Teklad ⅛″ corncob bedding 7902 inside bioBubble® Clean Rooms that provide H.E.P.A.filtered air into the bubble environment at 100 complete air changes perhour. All treatments and infectious challenges are carried out in a BSL2surgical suite. The environment is controlled to a temperature range of74°±4° F. and a humidity range of 30-70%. Treatment groups areidentified by cage card. All procedures carried out in this experimentare conducted in compliance with the laws, regulations, and guidelinesof the National Institutes of Health and with the approval of theTransPharm Animal Care and Use Committee.

Bacterial Cultures

The bacterial strain used in this study is a methicillin resistantStaphylococcus aureus strain USA300 (TPPS 1056), procured from thelaboratory of Barry Kreiswirth (Public Health Research Institute Center,New Jersey Medical School).

Skin Preparation

On Day 0 immediately prior to infection, each mouse is anesthetizedusing an isoflurane induction chamber and the dorsal area received 7-10applications and removals of Nexcare™ (3M) surgical tape in order toremove the outermost epidermal layer of skin.

Challenge

The organism is grown overnight at 37° C. in ambient atmosphere ontrypticase soy agar plates supplemented with 5% sheep blood cells. Onthe day of challenge (Day 0), cultures are aseptically swabbed andtransferred to culture tubes of TSB. The cultures are grown in a 37° C.water bath until the optical density reaches approximately 0.65 at 600nm, providing an undiluted bacterial concentration of approximately 109colony forming units (CFU)/mL. The cultures are then diluted to providea challenge inoculate of 6.0 log₁₀ CFU per mouse in a volume of 100 μL.Inoculum count is estimated before inoculation by optical density andconfirmed after inoculation by dilution and back count.

Immediately following tape stripping, each animal receives a topicalapplication with approximately 6.0 log₁₀ CFU per mouse in a volume of100 μL spread in an area approximately 0.75″×0.75″. Challenge is allowedto dry slightly before mice are brought out of anesthesia. The final CFUcount from the challenge suspension determined that 5.6 log₁₀ CFU permouse is delivered. Instillation of the bacterial challenge constitutestime 0 hour for the study.

Formulation and Dosing

A 10% (100 mg/mL) solution of Nu-3 is prepared in a fatty alcohol-basedgel. Hydrochloric acid is used to adjust the solution to pH 1.5.

Four hours following challenge, mice receive a topical application oftest article or vehicle gel to the dorsal area previously stripped andinoculated with bacteria. The gel is spread evenly using a sterile loop.Control animals do not receive treatment. Mice are housed individuallyfollowing treatment until time of harvest.

Endpoint Analysis

Mice are harvested at designated time points based upon the study. Fourmice are harvested from each group at each time point.

Following euthanasia, a section of excised skin approximately 0.5″×0.5″is aseptically removed from the infected/treated area and transferred tovials with 2.0 mL of sterile water and weighed. Tissues are allowed toset at room temperature for 10 minutes. Tissue pH is measured usinglitmus paper and recorded. Following pH measurement, tissues arehomogenized using a bead beater. The homogenate is serially diluted fromneat to 10-7 in PBS and plated in duplicate 5 μL spots onto trypticasesoy agar plates supplemented with 5% sheep blood cells. 100 μL of theundiluted (neat) homogenate is plated for each sample. Plates areincubated overnight at 37° C. in ambient atmosphere. Colony formingunits (CFU) are tabulated for each treatment per gram of tissue.

Results and Discussion

None of the mice displayed any acute adverse events associated with thetreatments. None of the mice succumbed to the infection or showed signsof morbidity, which could be attributed to penetration of the infectioninto the circulatory system or deep tissue. No groups displayed adversesigns beyond those expected for mice which have received a superficialbacterial infection.

The baseline bacterial burden of the untreated group (Group 1) is 6.94log₁₀ CFU at 5 hours post-challenge and 8.14 log₁₀ CFU at 12 hourspost-challenge. Group 2, which was administered vehicle, did notdemonstrate significant changes when compared to the untreated group.Group 3, which received Nu-3, showed significant decreases in CFU burdenat 5 and 12 hours post-challenge when compared to that of untreatedcontrol animals. Group 3 also showed significant decreases in CFU burdenat all harvest time points when compared to vehicle-treated animals.

Together, these data demonstrate that Staphylococcus aureus establishesa robust dermal infection. Topical administration of Nu-3 significantlydecreases bacterial burden at 5 and 12 hours post-challenge whencompared to untreated controls and at all time points when compared tothe vehicle-treated group.

While embodiments have been disclosed hereinabove, the present inventionis not limited to the disclosed embodiments. Instead, this applicationis intended to cover any variations, uses, or adaptations of theinvention using its general principles. Further, this application isintended to cover such departures from the present disclosure as comewithin known or customary practice in the art to which this inventionpertains and which fall within the limits of the appended claims.

1-35. (canceled)
 36. A method of treating an infection in a patient inneed thereof, the method comprising administering to the patient aneffective amount of a formulation comprising: from about 1% to about 20%(weight/weight) of a Nu-3 having the formula:

or a pharmaceutically acceptable salt thereof; from about 1% to about10% (weight/weight) of a fatty alcohol thickening agent; from about 0.5%to about 5% (weight/weight) of a nonionic polymer emulsifier; and fromabout 65% to about 97.5% (weight/weight) of a diluent.
 37. The method ofclaim 36, wherein the patient is administered at least one additionalactive ingredient.
 38. The method of claim 36, wherein the patient is ahuman.
 39. The method of claim 36, wherein the fatty alcohol thickeningagent is selected from the group consisting of cetyl alcohol, laurylalcohol, stearyl alcohol, cetostearyl alcohol, myristyl alcohol,docosanol alcohol, oleyl alcohol, and combinations thereof.
 40. Themethod of claim 36, wherein the nonionic polymer is selected from thegroup consisting polysorbate 20, polysorbate 60, polysorbate 80,ceteareth-12, ceteareth-20, ceteareth-30, ceteth-10, ceteth-20,steareth-10, steareth-20, steareth-40, steareth-100, and combinationsthereof.
 41. The method of claim 36, wherein the diluent is selectedfrom the group consisting of water, glycerol, mannitol, saline,phosphate buffered saline, and combinations thereof.
 42. The method ofclaim 36, wherein the administration is topical administration.
 43. Themethod of claim 36, wherein the Nu-3, or a pharmaceutically acceptablesalt thereof, is present in the formulation in an amount from about 1%to about 15% (weight/weight).
 44. The method of claim 36, wherein thefatty alcohol thickening agent is present in the formulation in anamount from about 2% to about 8% (weight/weight).
 45. The method ofclaim 36, wherein the nonionic polymer emulsifier is present in theformulation in an amount greater than about 0.5% (weight/weight). 46.The method of claim 36, further comprising from about 1% to about 10%(weight/weight) of an acid selected from the group consisting ofhydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid,carbonic acid, sulfuric acid, and phosphoric acid.
 47. The method ofclaim 36, wherein the Nu-3, or a pharmaceutically acceptable saltthereof, is administered to the patient at a dose from about 1 to about500 mg of Nu-3, or a pharmaceutically acceptable salt thereof, per kg ofpatient body weight.
 48. The method of claim 36, wherein theadministration is carried out as a multiple dose regimen.
 49. The methodof claim 36, wherein the administration is carried out one or more timesper day.
 50. The method of claim 36, wherein the formulation comprises:from about 1% to about 20% (weight/weight) of the Nu-3, or apharmaceutically acceptable salt thereof; from about 1% to about 10%(weight/weight) of a fatty alcohol thickening agent selected from thegroup consisting of cetyl alcohol, lauryl alcohol, stearyl alcohol,cetostearyl alcohol, myristyl alcohol, docosanol alcohol, oleyl alcohol,and combinations thereof; from about 0.5% to about 5% (weight/weight) ofa nonionic polymer emulsifier selected from the group consisting ofpolysorbate 20, polysorbate 60, polysorbate 80, ceteareth-12,ceteareth-20, ceteareth-30, ceteth-10, ceteth-20, steareth-10,steareth-20, steareth-40, steareth-100, and combinations thereof; andfrom about 65% to about 97.5% (weight/weight) of a diluent selected fromthe group consisting of water, glycerol, mannitol, saline, phosphatebuffered saline, and combinations thereof.
 51. The method of claim 36,wherein the infection is selected from the group consisting of aninfection of an ulcer, an infection of a wound, and an otitis externainfection.
 52. The method of claim 36, wherein the infection is selectedfrom the group consisting of acne vulgaris, onychomycosis,conjunctivitis, oral mucositis, an infection of a mucosal surface, avaginal infection, and combinations thereof.
 53. The method of claim 36,wherein the infection is an infection of a diabetic foot ulcer or acomplicated venous leg ulcer.
 54. The method of claim 36, wherein theinfection is an infection of a burn wound.
 55. The method of claim 36,wherein the infection is an infection of a wound or ulcer.